Animal cell culture is a basic technique in the fields of biology and medicine. The production of living cells in vitro, in the laboratory, permits numerous applications that would be difficult or impossible in vivo, in the living animal. The culture of animal cells requires a defined medium containing specific quantities of certain chemicals, and in addition for most cells, up to 15% of an undefined nutrient medium usually fetal bovine serum (FBS). Serum from newborn calves and other mammals is also used, but FBS is preferred because of its high level of growth factors and low cross-reactivity with other animal cells. FBS or other mammalian products are also used to coat the surface of culture-ware to promote cell attachment.
The production of FBS in this country is an estimated 700,000 liters annually worth $300 to $400 million. The industry obtains fetal calves for bleeding from slaughter houses, or in some cases, rears herds of cattle for this purpose. These herds are held in as isolated a situation as possible in order to prevent disease. Whole blood is obtained aseptically (by syringe) from an animal, centrifuged to separate cells from serum, and the serum filtered to 0.22 microns to remove most infections agents. Often, serum is heated to 56.degree. C. to inactivate the complement system, a group of immune proteins.
Insect cell culture has also been conducted for many years to develop control methods for this important animal group, and as a model for biological processes in humans and higher animals. More recently, insect cell culture and a virus vector have become a valuable tool for the expression of foreign genes. This technique is superior to the production of foreign proteins by bacteria as higher yields, better "copies" and more complex eukaryotic proteins can be obtained (Smith et al., 1983). Some examples of the recombinant proteins produced by insect cell expression systems are human interferon and interleukin-2, substances that are injected as therapy in human subjects.
An example of the current method of protein production is: 1. culture of an insect cell line (Spodoptera frugiperda), 2. insertion of the desired foreign gene in the baculovirus, Autographa Californica Polyhedral Virus (Ac NPV), 3. infection of the insect cell line with this virus, and 4. extraction of the resulting foreign protein from the infected insect cells.
Contamination of cell cultures because of infectious organisms in serum can be a serious problem. Bacteria, fungi, viruses and mycoplasma have been isolated from bovine serum. A decade ago mycoplasma from bovine serum was the second major group of contaminants found in cell culture (Barile, 1977). Now, animal sera are routinely screened for mycoplasma, viruses and other known contaminants. However, a more serious cause for concern is an all-protein infectious agent called a prion for which no test is available (Prusiner, 1982). This prion causes a fatal brain disease in mammals called Bovine Spongiform Encephalopathy (BSE) or "mad cow disease." BSE occurs in sheep, cows, and other mammals, and is most likely the cause of similar neuro-degenerative diseases such as such as Creutzfeld-Jakob disease in humans. In Britain since 1986, mad cow disease has resulted in the destruction of over 100,000 cattle and fears for contamination of the meat supply or other animal products. The disease has also turned up in cattle in many other countries. Consequently, serum from these countries cannot be imported for use in the U.S.
During insect cell culture procedures, most insect cells are maintained in a defined media plus 10% FBS. Therefore any infectious agent in the FBS could contaminate a recombinant protein made by the cells, and could be transmitted to humans receiving this protein as therapy.
The method of the present invention is especially timely as BSE has recently been found in Canadian cattle (Campbell, 1993) and is strongly implicated as the cause of death in Wisconsin mink which were fed protein meal made from dairy cow carcasses (Marsh, 1993). In Europe there is evidence of the disease in humans receiving recombinant human growth hormone contaminated with infected bovine serum (Knauer, 1993).